Muscle atrophy inhibitor

ABSTRACT

An extract of  Citrus depressa , preferably an organic solvent extract of a fruit and/or leaf of  Citrus depressa , a supercritical extract of a fruit and/or leaf of  Citrus depressa , or a subcritical extract of a fruit and/or leaf of  Citrus depressa , containing 0.3 mass % or more of a polymethoxyflavonoid in terms of solid matter, for example, 0.2 mass % or more of nobiletin and/or 0.1 mass % or more of tangeretin in terms of solid matter, or a polymethoxyflavonoid, such as nobiletin and tangeretin, is used as an active ingredient of a muscle atrophy inhibitor.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 14/365,796, filed Jun. 16, 2014 which is the U.S. National Phaseunder 35 U.S.C. §371 of International Application PCT/JP2012/083719,filed Dec. 26, 2012, which was published in a non-English language,which claims priority to JP 2011-283004, filed Dec. 26, 2011.

TECHNICAL FIELD

The present invention relates to a muscle atrophy inhibitor. A muscleatrophy inhibitor can be used as a drug, food, and feed.

BACKGROUND ART

Muscle atrophy refers to a state that muscle mass decreases due toreduction of muscle fiber number and reduction of muscle fiber volume,and is usually accompanied by decreased muscle force. Althoughappropriate exercise is effective for prophylaxis or improvement ofmuscle atrophy, recovery by exercise is difficult for sick persons andold people. Therefore, development of a drug or food effective forsuppression or improvement of muscle atrophy is expected.

As techniques for inhibiting muscle atrophy using ingredients containedin plants, there are known muscle atrophy inhibitors containingAraliaceae ginseng radix (Patent document 1), α-glycosylated hesperidin(Patent document 2), or stigmasterol (Patent document 3) as an activeingredient, and an inhibitor of muscle fiber type shift containing afruit-derived polyphenol (Patent document 4) as an active ingredient.

Further, as techniques for improving muscle function or suppressingreduction of muscle function, there are known a muscle functionreduction inhibitor containing catechins as an active ingredient (Patentdocument 5), a endurance improver containing resveratrol and/or grapeleaf extract as an active ingredient (Patent document 6), ananti-amyotrophic lateral sclerosis (ALS) agent containing rosmarinicacid or carnosic acid as an active ingredient (Patent document 7), andan anti-ALS agent containing a rosemary extract or sage extract as anactive ingredient (Patent document 8).

As a plant-derived health food raw material, extract of Citrus depressaattracts attention. Various efficacies of polymethoxyflavonoids such asnobiletin and tangeretin contained in extract of Citrus depressa havebeen found to date. For example, it is reported that nobiletin has aneurite outgrowth action (Patent document 9), anti-hypertension andanti-cancer actions (Patent document 10), heart disease preventing andtreating actions (Patent document 11), antiulcer action (Patent document12), and so forth. Further, it has been reported thatpolymethoxyflavonoids such as tangeretin and nobiletin have aneovascularization suppressing action (Patent document 13). Furthermore,it is known that flavonoids contained in Citrus species such as Citrusdepressa have a blood pressure elevation suppressing action (Patentdocument 14).

Further, the aforementioned inhibitor of muscle fiber type shift (Patentdocument 4) uses a polyphenol such as, specifically, procyanidincontained in fruits of Rosaceae plants such as apple, as an activeingredient.

However, such flavonoids as mentioned above are scarcely contained infruit juice, but are mostly contained in pericarps. Therefore, only bysqueezing fruits, these flavonoids are obtained only at a low content.

Polymethoxyflavonoids constitute one class of flavonoid, have a specialstructure in which a plurality of phenolic hydroxyl groups aremethylated, and are mainly contained in Citrus species. It has also beenreported that polymethoxyflavonoids such as nobiletin or tangeretin aremetabolized in the liver after intake, and the generated metabolitesenhance anti-inflammatory action. For example, methoxy groups ofnobiletin are converted into hydroxyl groups by metabolism in the liverof rat, and nobiletin derivatives having 4′-OH, 7-OH, 6-OH, 3′,4′-diOH,6,7-diOH or the like are generated as metabolites. Further, it has beenreported that, from tangeretin, tangeretin derivatives having 4′-OH,3′,4′-diOH, 7,4′-diOH, 6,7-diOH or the like are generated as metabolites(Non-patent document 1).

Although several plant-derived ingredients having a muscle atrophyinhibition action are known as described above, it is not known thatextract of Citrus depressa or a polymethoxyflavonoid such as nobiletinand tangeretin has a muscle atrophy inhibition action.

As a method for preparing a muscle atrophy model for evaluating foodmaterials, a method using a glucocorticoid, and a method using hindlimbimmobilization or unloading are known. There have been reported thateffect of a branched chain amino acid on cross-sectional areas of musclefibers etc. (Non-patent document 2), and effects of creatine (Non-patentdocument 3) and vitamin E (Non-patent document 4) on muscle weight wereevaluated by using a muscle atrophy model derived with a glucocorticoid.Further, there have also been reported that effects of resveratrol(Non-patent document 5) and fish oil (Non-patent document 6) on muscleweight were evaluated by using a muscle atrophy model prepared by usinghindlimb immobilization or unloading.

However, there is no report concerning evaluation of muscle atrophyinhibition action of extract of Citrus depressa or ingredients thereofwith these models or others.

PRIOR ART REFERENCES Patent Documents

-   Patent document 1: Japanese Patent Laid-open (Kokai) No. 2008-179620-   Patent document 2: Japanese Patent Laid-open No. 2009-7313-   Patent document 3: Japanese Patent Laid-open No. 2010-47529-   Patent document 4: Japanese Patent Laid-open No. 2006-328031-   Patent document 5: Japanese Patent Laid-open No. 2008-13473-   Patent document 6: Japanese Patent Laid-open No. 2007-145809-   Patent document 7: Japanese Patent Laid-open No. 2009-256282-   Patent document 8: Japanese Patent Laid-open No. 2009-256283-   Patent document 9: Japanese Patent No. 4633897-   Patent document 10: International Patent Publication WO2006/49234-   Patent document 11: Japanese Patent Laid-open No. 2011-37798-   Patent document 12: Japanese Patent Laid-open No. 6-72870-   Patent document 13: Japanese Patent Laid-open No. 2004-83417-   Patent document 14: Japanese Patent Laid-open No. 2001-240539

Non-Patent Documents

-   Non-patent document 1: Koga, N. et al., Biol. Pharm. Bull. 30(12),    2317-2323, 2007-   Non-patent document 2: Yamamoto, D. et al., Muscle & Nerve,    41:819-827, 2010-   Non-patent document 3: Menezes, L. G. et al., J. Appl. Physiol.,    102:698-703, 2007-   Non-patent document 4: Ohtsuka, A. et al., J. Nutr. Sci. Vitaminol.,    44, 779-786, 1998-   Non-patent document 5: Jackson, J. R. et al., Am. J. Physiol.    Integr. Comp. Physiol., 299:R1572-R1581, 2010-   Non-patent document 6: You, J.-S. et al., Appl. Physiol. Nutr.    Metab., 35:310-318, 2010

SUMMARY OF THE INVENTION Object to be Achieved by the Invention

An object of the present invention is to provide a muscle atrophyinhibitor that can be safely ingested, and a food or drink containingit.

Means for Achieving the Object

The inventors of the present invention conducted various researches inorder to achieve the aforementioned object. As a result, they found thatan extract of Citrus depressa or an ingredient thereof had a superiormuscle atrophy inhibition action, and accomplished the presentinvention.

The present invention thus provides the following.

[1] A muscle atrophy inhibitor comprising an extract of Citrus depressaas an active ingredient.[2] The muscle atrophy inhibitor according to [1], wherein the extractof Citrus depressa is an organic solvent extract of a fruit and/or leafof Citrus depressa.[3] The muscle atrophy inhibitor according to [1], wherein the extractof Citrus depressa is a supercritical extract or subcritical extract ofa fruit and/or leaf of Citrus depressa.[4] The muscle atrophy inhibitor according to [2], wherein the organicsolvent is selected from the group consisting of methanol, ethanol,propanol, butanol, ethyl acetate, acetone, hexane, chloroform, diethylether, and these organic solvents comprising water.[5] The muscle atrophy inhibitor according to [4], wherein the organicsolvent is ethanol or water-comprising ethanol.[6] The muscle atrophy inhibitor according to [1], wherein the extractof Citrus depressa comprises polymethoxyflavonoid.[7] The muscle atrophy inhibitor according to [6], wherein the extractof Citrus depressa comprises 0.3 mass % or more of apolymethoxyflavonoid in terms of solid matter.[8] The muscle atrophy inhibitor according to [6], wherein thepolymethoxyflavonoid comprises nobiletin and/or tangeretin.[9] The muscle atrophy inhibitor according to [8], wherein the extractof Citrus depressa comprises 0.2 mass % or more of nobiletin and/or 0.1mass % or more of tangeretin in terms of solid matter.[10] The muscle atrophy inhibitor according to [6], which furthercomprises a clathrating agent for making the polymethoxyflavonoidwater-soluble.[11] The muscle atrophy inhibitor according to [10], wherein theclathrating agent is cyclodextrin, and content thereof is 0.1 to 95 mass% based on the total mass of solid matter of the extract of Citrusdepressa and the cyclodextrin.[12] A muscle atrophy inhibitor comprising a polymethoxyflavonoid as anactive ingredient.[13] The muscle atrophy inhibitor according to [12], wherein thepolymethoxyflavonoid comprises nobiletin and/or tangeretin.[14] A food or drink comprising 0.3 mass % or more of the muscle atrophyinhibitor according to [6] as polymethoxyflavonoid content in terms ofsolid matter.[15] A food or drink comprising 0.2 mass % or more of the muscle atrophyinhibitor according to [13] as nobiletin content in terms of solidmatter.[16] A food or drink comprising 0.1 mass % or more of the muscle atrophyinhibitor according to [13] as tangeretin content in terms of solidmatter.[17] An extract of Citrus depressa to be used for inhibiting muscleatrophy.[18] A polymethoxyflavonoid to be used for inhibiting muscle atrophy.[19] A method for inhibiting muscle atrophy, which comprisesadministering an extract of Citrus depressa to a mammal.[20] The method for inhibiting muscle atrophy according to [19], whereinthe extract of Citrus depressa comprises polymethoxyflavonoid.[21] The method for inhibiting muscle atrophy according to [19], whereinthe extract of Citrus depressa comprises 0.3 mass % or more of apolymethoxyflavonoid in terms of solid matter.[22] The method for inhibiting muscle atrophy according to [20], whereinthe polymethoxyflavonoid comprises nobiletin and/or tangeretin.[23] The method for inhibiting muscle atrophy according to [22], whereinthe extract of Citrus depressa comprises 0.2 mass % or more of nobiletinand/or 0.1 mass % or more of tangeretin in terms of solid matter.[24] A method for inhibiting muscle atrophy, which comprisesadministering a polymethoxyflavonoid to a mammal.[25] The method for inhibiting muscle atrophy according to [24], whereinthe polymethoxyflavonoid comprises nobiletin and/or tangeretin.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 includes graphs showing change of expression of muscleatrophy-related genes provided by administration of an extract of Citrusdepressa.

CTL: Dexamethasone non-administration group

DEX: Dexamethasone administration group

SE: Dexamethasone and extract of Citrus depressa (shiikuwasha extract)administration group

FIG. 2 includes graphs showing change of expression of muscleatrophy-related genes provided by administration of polymethoxyflavonoid(PMF), nobiletin

(NOB), and tangeretin (TAN).

CTL: Dexamethasone non-administration group

DEX: Dexamethasone administration group

PMF: Dexamethasone and polymethoxyflavonoid administration group

NOB: Dexamethasone and nobiletin administration group

TAN: Dexamethasone and tangeretin administration group

FIG. 3 is a graph showing suppression of reduction of soleus muscleweight in hindlimb-immobilized rats provided by administration ofextract of Citrus depressa.

CTL: Non-treated group

FIX: Hindlimb immobilization group

SE: Hindlimb immobilization and extract of Citrus depressa (shiikuwashaextract) administration group

EMBODIMENTS FOR CARRYING OUT THE INVENTION

Hereafter, preferred embodiments of the present invention will beexplained in detail. However, the present invention is not limited tothe following preferred embodiments, but can be freely modified withinthe scope of the present invention.

The muscle atrophy inhibitor of the present invention contains anextract of Citrus depressa or a polymethoxyflavonoid as an activeingredient. Examples of the polymethoxyflavonoid includepolymethoxyflavonoids contained in Citrus depressa or an extractthereof. The polymethoxyflavonoids contained in Citrus depressa or anextract thereof have a structure generally represented by the followingformula, and specific examples include nobiletin, tangeretin,5-demethylated nobiletin, 8-demethoxylated nobiletin (sinensetin),6-demethoxylated tangeretin, 6-demethoxylated nobiletin, citromitin,5,6,7,8,4-pentamethoxyflavanone, and so forth. Among these, nobiletinand tangeretin are preferred. In the following chemical formula, R, R₁,R₂, R₃, and R₄ are OMe, OMe, H, Me, and OMe, respectively, in nobiletin,or OMe, H, H, Me, and OMe, respectively, in tangeretin (Me representsmethyl group, OMe represents methoxy group, and H represents hydrogen).

The polymethoxyflavonoid may consist of a single kind ofpolymethoxyflavonoid, or a mixture of arbitrary two or more kinds ofpolymethoxyflavonoids.

(In the formula, R, R₁, R₂, and R₄ independently represent hydrogen atomor methoxy group, and R₃ represents hydrogen atom or methyl group.)

The polymethoxyflavonoid may be extracted from a fruit, leaf, root, stemetc. of a Citrus species or another plant containing that substance, ormay be produced by chemical synthesis. As nobiletin and tangeretinobtained by chemical synthesis, commercial synthetic products (forexample, those produced by Tokyo Chemical Industry), and so forth can beused.

The extract of Citrus depressa can be produced by, for example,extraction of fruit and/or leaf of Citrus depressa with water and/or anorganic solvent. The organic solvent may contain water. Citrusdepressa(Shiikuwasha) is a kind of Citrus species belonging to thefamily Rutaceae.

The fruit and/or leaf may be the whole fruit and/or leaf, or may be apart thereof. For example, the fruit may be pulp or pericarp. Further,the fruit and/or leaf may be used as they are, or may be used aftercrushing. Further, the fruit and/or leaf may be a juice extractionresidue of a fruit and/or leaf, or a part thereof. Hereafter, thesefruit and/or leaf, a part thereof, crushed product thereof, and juiceextraction residue thereof may be referred to as “Citrus depressa fruitand/or leaf etc.”

Examples of the organic solvent include methanol, ethanol, propanol,butanol, ethyl acetate, acetone, hexane, chloroform, diethyl ether,these organic solvents containing water, combination of each of theseorganic solvents and these organic solvents containing water, but amongthese, ethanol is preferred. Although water content in the organicsolvent is not particularly limited, it is preferably 0 to 90 mass %,more preferably 0 to 40 mass %.

Although amount of the organic solvent with respect to the Citrusdepressa fruit and/or leaf etc. used in the extraction with the organicsolvent is not particularly limited, ratio (weight ratio) of the Citrusdepressa fruit and/or leaf etc.:organic solvent is preferably 1:1 to1:100, more preferably 1:1 to 1:20.

Although the method of the extraction is not particularly limited,examples include, for example, a method of adding an organic solvent toCitrus depressa fruit and/or leaf etc., performing extraction preferablyfor 5 minutes to 3 hours, and then collecting the liquid phase by asolid/liquid separation means such as filtration or centrifugalseparation.

Further, after the extraction or before drying process, it is preferableto add a clathrating agent for making the polymethoxyflavonoidwater-soluble. If such a clathrating agent is used, effects of improvingwater solubility, digestion and absorption, and flavor of thepolymethoxyflavonoid can be expected. As the clathrating agent, it ispreferable to use a compound for clathrate such as cyclodextrin. In thecase of cyclodextrin, amount of the clathrating agent is preferably 0.1to 95 mass %, preferably 1 to 90 mass %, based on the total mass of thesolid matter of the extract of Citrus depressa and cyclodextrin.

The extract of Citrus depressa of the present invention can also beproduced by supercritical extraction. Specifically, it can be producedby, for example, subjecting frozen and crushed Citrus depressa fruit orleaf, or Citrus depressa fruit or leaf powdered by lyophilization or hotair-drying to supercritical extraction performed under the followingconditions (a) to (d).

(a) Extraction solvent is carbon dioxide (carbon dioxide gas).(b) Extraction temperature is 25 to 120° C.

(c) Pressure is 5.5 to 60 MPa.

(d) Extraction time is 5 to 70 minutes.

As the extraction fluid, it is possible to use supercritical propane,supercritical ethylene, supercritical 1,1,1,2-tetrafluoroethane, or thelike, in order to improve extraction efficiency of the Citrus depressafruit or leaf. However, in order to increase safety as food or drink, itis preferable to use carbon dioxide (carbon dioxide gas). The extractiontemperature may be appropriately chosen to be in the temperature rangeof 31.1 to 120° C., but in order to improve the extraction efficiencyand increase the content of the polymethoxyflavonoid, especiallynobiletin and/or tangeretin, it is preferably in the range of 40 to 80°C., more preferably in the range of 60 to 80° C. The pressure ispreferably in the range of 5.5 to 60 MPa, more preferably in the rangeof 20 to 40 MPa. Further, in the present invention, ethanol, water, orthe like may be used as an entrainer, in order to improve the extractionefficiency. Although the extraction time may be appropriately chosenaccording to the temperature or pressure, it is, for example, preferablyin the range of 10 to 50 minutes, more preferably 20 to 30 minutes.

The extraction operation can be performed by using a commerciallyavailable apparatus.

The extract of Citrus depressa of the present invention can also beproduced by subcritical extraction. Specifically, it can be produced by,for example, subjecting frozen and crushed Citrus depressa fruit orleaf, or Citrus depressa fruit or leaf powdered by lyophilization or hotair-drying to subcritical extraction performed under the followingconditions (a) to (d).

(a) Extraction solvent is water.(b) Extraction temperature is 140 to 200° C.

(c) Pressure is 3 to 15 MPa.

(d) Extraction time is 0 to 10 minutes.

The extraction time of 0 minute means that immediately after thetemperature is raised to the objective extraction temperature from thestart of the extraction, the temperature is lowered by cooling to thelevel at the start of the extraction.

Examples of the extraction fluid used for the subcritical extractioninclude, for example, water and carbon dioxide. However, in order toincrease safety as food or drink, it is preferable to use water.

In the case of using water as the extraction fluid, the extractiontemperature may be appropriately chosen to be in the temperature rangeof 140 to 200° C., but in order to improve the extraction efficiency andincrease the content of the polymethoxyflavonoid, especially nobiletinand/or tangeretin, it is preferably in the range of 140 to 180° C. Thepressure is preferably in the range of 3 to 15 MPa in the case of usingwater as the extraction fluid.

Although the extraction time may be appropriately chosen according tothe temperature or pressure, it is preferably in the range of 0 to 10minutes, more preferably 0 to 5 minutes.

The extraction operation can be performed by using a commerciallyavailable apparatus.

Yields of nobiletin and tangeretin in an extract obtained as describedabove by the extraction method using water, an organic solvent, or anorganic solvent containing water, supercritical extraction, orsubcritical extraction are usually about 0.001 to 3 mass %, and 0.0001to 2 mass %, respectively, based on the weight of Citrus depressa fruitand/or leaf etc.

The extract of Citrus depressa obtained as described above containspreferably 0.3 mass % or more, more preferably 0.6 mass % or more, stillmore preferably 1 mass % or more, further preferably 3 mass % or more,particularly preferably 10 mass % or more, of the polymethoxyflavonoidin terms of solid matter. Further, such a extract of Citrus depressacontains preferably 0.2 mass % or more, more preferably 0.4 mass % ormore, still more preferably 2 mass % or more, particularly preferably 5mass % or more, of nobiletin, and/or preferably 0.1 mass % or more, morepreferably 0.2 mass % or more, still more preferably 1 mass % or more,particularly preferably 2 mass % or more, of tangeretin, in terms ofsolid matter. The phrase “in terms of solid matter” has the same meaningas “amount as solid matter (solid content)”. Further, expression that adrug, food or the like contains X % or more of the extract of Citrusdepressa, polymethoxyflavonoid, nobiletin, or tangeretin “in terms ofsolid matter” means that the ratio of the amount of the solid matter ofthe extract of Citrus depressa, polymethoxyflavonoid, nobiletin, ortangeretin to the amount of the solid matter of the drug, food or thelike is X %.

The extract may be used as it is, or may be used after concentration,and the solvent may be partially or completely removed. Theconcentration or removal of the solvent can be carried out by suchmethods as various chromatography techniques, distillation,solidification by drying, and recrystallization. In particular, organicsolvents not preferred to be contained in a drug or food or drink, forexample, methanol, propanol, butanol, ethyl acetate, acetone, hexane,chloroform, diethyl ether, etc., is preferably removed. Further, inorder to increase the content of the polymethoxyflavonoid, especiallynobiletin and/or tangeretin, the extract may be fractionated. Thecontent of the polymethoxyflavonoid such as nobiletin and/or tangeretincan be measured by HPLC or the like.

Such an extract of Citrus depressa or polymethoxyflavonoid as describedabove can be used as it is as an active ingredient of the muscle atrophyinhibitor (henceforth also referred to as “agent of the presentinvention”), food, drink, or feed. The extract of Citrus depressa orpolymethoxyflavonoid may be in the form of a solution, or it may belyophilized or spray-dried in a conventional manner, then stored andused as powder.

The agent of the present invention can be used as a drug or an activeingredient thereof as one embodiment. As the agent of the presentinvention, an extract of Citrus depressa or polymethoxyflavonoid can beorally administered as it is or as a combination with a pharmaceuticallyacceptable carrier to a mammal including human.

Preparation form of the agent of the present invention is notparticularly limited, and examples include tablets (includingsugar-coated tablets, enteric coated tablets, and buccal tablets),powders, capsules (including enteric capsules and soft capsules),granules (including coated granules), pills, troches, enclosed liposomeagents, solutions, pharmaceutically acceptable sustained releasepreparations of these, and so forth. When the preparation is prepared,additives commonly used in usual oral drugs as pharmaceuticalingredients, such as carrier, excipient, binder, disintegrating agent,lubricant, stabilizer, flavoring agent, diluent, surfactant and solvent,can be used. Further, so long as the effect of the present invention isnot degraded, the extract of Citrus depressa or polymethoxyflavonoid maybe used together with an agent or pharmaceutical composition having amuscle atrophy inhibition action, which is already known or will befound in future. The pharmaceutical composition used together may becontained in the agent of the present invention as one of activeingredients, or may not be contained in the agent of the presentinvention, but combined as a separate drug with the agent of the presentinvention to form a commercial product.

Examples of the carrier and excipient used for the aforementionedpreparation include lactose, glucose, sucrose, mannitol, potato starch,corn starch, calcium carbonate, calcium phosphate, calcium sulfate,crystalline cellulose, glycyrrhizae radix pulverata, gentianae radixpulverata, and so forth, and examples of the binder include, forexample, starch, gelatin, syrup, polyvinyl alcohol, polyvinyl ether,polyvinylpyrrolidone, hydroxypropylcellulose, ethylcellulose,methylcellulose, carboxymethylcellulose, and so forth.

Examples of the disintegrating agent include starch, agar, gelatinpowder, sodium carboxymethylcellulose, calcium carboxymethylcellulose,crystalline cellulose, calcium carbonate, sodium hydrogencarbonate,sodium arginate, and so forth.

Examples of the lubricant include magnesium stearate, hydrogenatedvegetable oil, Macrogol, and so forth, and examples of the colorantinclude Red No. 2, Yellow No. 4, Blue No. 1, which are allowed to beadded to drugs, and so forth.

Tablets and granules can be coated with sucrose, hydroxypropylcellulose,purified shellac, gelatin, sorbitol, glycerol, ethylcellulose,hydroxypropylcellulose, hydroxypropylmethylcellulose,polyvinylpyrrolidone, cellulose acetate phthalate,hydroxypropylmethylcellulose phthalate, methyl methacrylate, methacrylicacid polymer, and so forth, as required.

One aspect of the present invention is use of an extract of Citrusdepressa or polymethoxyflavonoid in preparation of a drug for inhibitingmuscle atrophy. Another aspect of the present invention is an extract ofCitrus depressa or polymethoxyflavonoid to be used for inhibiting muscleatrophy. Still another aspect of the present invention is a method forinhibiting muscle atrophy comprising administering an extract of Citrusdepressa or polymethoxyflavonoid to a mammal.

Although amount of the extract of Citrus depressa orpolymethoxyflavonoid contained in the agent of the present invention isnot particularly limited and can be appropriately chosen, when anextract of Citrus depressa is used, for example, the amount ispreferably 1 mass % or more, more preferably 10 mass % or more, in termsof the amount of the solid matter contained in the extract of Citrusdepressa. Alternatively, the amount of the extract of Citrus depressacontained in the agent of the present invention is preferably 0.3 mass %or more, more preferably 0.6 mass % or more, further preferably 3.0 mass% or more, particularly preferably 10 mass % or more, in terms ofpolymethoxyflavonoid content. Although the maximum content of theextract of Citrus depressa is not particularly limited, it may be, forexample, 95 mass % or less, 90 mass % or less, or 50 mass % or less, interms of the amount of the solid matter in the extract of Citrusdepressa, or it may be, for example, 95 mass % or less, 80 mass % orless, 60 mass % or less, or 40 mass % or less, in terms of the amount ofpolymethoxyflavonoid.

Further, when the polymethoxyflavonoid is used, the amount ofpolymethoxyflavonoid contained in the agent may be 0.001 mass % or more,preferably 0.1 mass % or more, more preferably 0.3 mass % or more,further preferably 0.6 mass % or more, still further preferably 3.0 mass% or more, particularly preferably 10 mass % or more, in terms of solidmatter. Although the maximum content of the polymethoxyflavonoid is notparticularly limited, it may be, for example, 95 mass % or less, 70 mass% or less, 60 mass % or less, 50 mass % or less, or 40 mass % or less.

When nobiletin is used as the polymethoxyflavonoid, the amount ofnobiletin contained in the agent may be 0.0007 mass % or more,preferably 0.07 mass % or more, still more preferably 0.2 mass % ormore, further preferably 0.4 mass % or more, still further preferably2.0 mass % or more, particularly preferably 5 mass % or more, in termsof solid matter. Although the maximum content of nobiletin is notparticularly limited, it may be, for example, 95 mass % or less, 70 mass% or less, 50 mass % or less, 30 mass % or less, or 10 mass % or less.

When tangeretin is used as the polymethoxyflavonoid, the amount oftangeretin contained in the agent may be 0.0004 mass % or more,preferably 0.04 mass % or more, still more preferably 0.1 mass % ormore, further preferably 0.2 mass % or more, still further preferably1.0 mass % or more, particularly preferably 2 mass % or more, in termsof solid matter. Although the maximum content of nobiletin is notparticularly limited, it may be, for example, 95 mass % or less, 70 mass% or less, 50 mass % or less, 30 mass % or less, or 10 mass % or less.

When two or more kinds of polymethoxyflavonoids, such as nobiletin,tangeretin, and other polymethoxyflavonoids, are used, the contentthereof in the agent may be appropriately chosen to be within theaforementioned ranges.

The agent of the present invention is useful for prophylactic andtherapeutic treatments of muscle atrophy, for example, muscle atrophyresulting from aging, bedridden, sedentary lifestyle, or spaceflight;muscle atrophy resulting from immobilization of limbs performed fortreatment of injury etc., or postoperative rest; muscle atrophyresulting from side reactions of drugs such as steroids; and muscleatrophy resulting from paralysis, spinal cord injury, traumatic injuryof peripheral nerve, osteoarthritis, rheumatoid arthritis, diabetes,thermal burn, polio, Guillain-Barre syndrome, muscular dystrophy,congenital myotonia, infectious disease accompanied by inflammation suchas AIDS and viral hepatitis, sepsis accompanying infectious disease,inflammatory bowel disease, connective tissue disease, renal failure,hepatic failure, cardiac failure, cancer, malignant tumor, cachexia,anorexia or hypercatabolism in terminal symptoms of a disease, and soforth; reduction of muscle force resulting from muscle atrophy; andamyotrophic lateral sclerosis and recovery of activities of daily living(ADL) at the time of rehabilitation. Further, the prophylactic andtherapeutic treatments of muscle atrophy include suppressing expressionof a muscle atrophy-related gene (atrogene) and/or a gene thatparticipates in suppression of muscle growth. Examples of the muscleatrophy-related gene include MuRF1 (Nikawa, T. et al., The FASEB Journalexpress article 10.1096/fj.03-0419fje. Published online, Jan. 8, 2004),and atrogin-1 (Gomes, M. D. et al., PNAS, 98(25), 2001), and examples ofthe gene that participates in suppression of muscle growth includemyostatin (also called growth/differentiation factor 8) gene.

Time for administration of the agent of the present invention is notparticularly limited, and can be appropriately chosen according to astate of an object of the administration.

Dose of the agent of the present invention is appropriately chosendepending on age, sex, state of the object of administration, otherconditions, and so forth. The dose is preferably chosen to be in therange of 1 to 250 mg/kg/day as a standard in terms of the amount of thesolid matter contained in the extract of Citrus depressa.

The agent can be administered at a dose of preferably 0.03 mg/kg/day ormore, more preferably 0.3 mg/kg/day or more, further preferably 3mg/kg/day or more, particularly preferably 30 mg/kg/day or more, as astandard in terms of the amount of polymethoxyflavonoid contained in thesolid matter of the extract of Citrus depressa. In this case, themaximum dose may be 150 mg/kg/day or less, preferably 120 mg/kg/day orless, more preferably 90 mg/kg/day or less, particularly preferably 60mg/kg/day or less.

The dose of the agent of the present invention in terms of the amount ofpolymethoxyflavonoid may be preferably 0.03 mg/kg/day or more, morepreferably 0.3 mg/kg/day or more, further preferably 3 mg/kg/day ormore, particularly preferably 30 mg/kg/day or more, as a standard. Themaximum dose in this case may be 150 mg/kg/day or less, preferably 120mg/kg/day or less, more preferably 90 mg/kg/day or less, particularlypreferably 60 mg/kg/day or less.

The dose in terms of the amount of nobiletin is preferably 0.02mg/kg/day or more, more preferably 0.2 mg/kg/day or more, furtherpreferably 2 mg/kg/day or more, particularly preferably 20 mg/kg/day ormore, as a standard. The maximum dose in this case may be 90 mg/kg/dayor less, preferably 72 mg/kg/day or less, more preferably 54 mg/kg/dayor less, particularly preferably 36 mg/kg/day or less.

The dose in terms of the amount of tangeretin may be preferably 0.01mg/kg/day or more, more preferably 0.1 mg/kg/day or more, furtherpreferably 1 mg/kg/day or more, particularly preferably 10 mg/kg/day ormore, as a standard. The maximum dose in this case may be 60 mg/kg/dayor less, preferably 48 mg/kg/day or less, more preferably 36 mg/kg/dayor less, particularly preferably 24 mg/kg/day or less.

When the administration period is long, for example, one to severalmonths or longer, the effect can be expected even with a dose of theagent of about 1/10 to 1/100 of the aforementioned ranges.

Regardless of the administration period, the daily dose of the agent canbe administered one time a day, or two or more times a day as dividedportions.

The agent of the present invention, or the extract of Citrus depressa orpolymethoxyflavonoid as the active ingredient of the agent may be addedto diets (drink or food).

Further, it is also possible to add the extract of Citrus depressa, thepolymethoxyflavonoid, or the agent of the present invention to a drinkor food as an active ingredient to produce a drink or food having amuscle atrophy inhibition action as one embodiment of the muscle atrophyinhibitor.

Forms and properties of the food and drink are not particularly limitedso long as the effect of the extract of Citrus depressa or thepolymethoxyflavonoid is not degraded, and they can be orally ingested,and they can be prepared by using usual raw materials used for foods anddrinks and usual methods, except that the extract of Citrus depressa orthe like is added.

Forms of such foods as mentioned above are not particularly limited, andthey may be in the form of liquid, paste, gellated solid, powder, or thelike. Examples include, for example, tablet confectioneries, and liquiddiets, as well as, for example, flour products such as bread, macaroni,spaghetti, noodles, cake mix, fry powder and bread crumbs; ready-to-eatfoods such as instant noodles, pot noodles, retort and cooked foods,canned cooking, foods for microwave heating, instant soup and stew,instant miso soup and Japanese clear soup, canned soup, freeze-driedfoods, and other ready-to-eat foods; processed agricultural productssuch as canned agricultural products, canned fruits, jams andmarmalades, pickles, cooked beans, dry agricultural products, andcereals (processed grain products); processed marine products such ascanned marine products, fish ham and sausages, seafood paste products,marine dainties, and tsukudani (marine products boiled in soy source;processed livestock products such as canned livestock products andpastes, and livestock meat ham and sausages; milks and dairy productssuch as processed milk, milk drinks, yoghurts, lactic acid drinks,cheese, ice creams, modified milk powders, creams, and other dairyproducts; oils and fats such as butter, margarines, and vegetable oils;basic seasoning such as soy sauce, miso, sauces, processed tomatoseasoning, mirin, and vinegars; complex seasonings and foods such ascooking mix, curry powder or roux, sauces for dipping, dressings, noodlesoups, spices, and other complex seasonings; frozen foods such as frozenfood materials, semi-cooked frozen foods, and cooked frozen foods;confectioneries such as caramel candies, candies, chewing gums,chocolates, cookies, biscuits, cakes, pies, snacks, crackers, Japanesesweets, rice confectioneries, bean confectioneries, dessert pastries,jellies, and other confectioneries; beverages such as carbonated drinks,natural fruit juices, fruit juice drinks, fruit juice soft drinks, fruitpulp drinks, fruit drinks with fruit pulp, vegetable based drinks, soymilk, soy milk drinks, coffee drinks, tea drinks, powdered drinks,concentrated drinks, sports drinks, nutritional beverage, alcoholicdrinks, and other beverages; other commercial foods such as baby foods,rice seasonings, and seaweed seasonings for boiled rice soaked with tea;modified milk powder for infants; enteral nutrients; functional foods(foods for specified health use, foods with nutrient function claims),and so forth.

Furthermore, by adding the extract of Citrus depressa, thepolymethoxyflavonoid, or the agent of the present invention to a feed asan active ingredient, a feed having a muscle atrophy inhibition actioncan be prepared, as one embodiment of the muscle atrophy inhibitor.

Form of the feed is not particularly limited. For example, the feed canbe prepared by blending cereals such as corn, wheat, barley, rye andmilo; vegetable oil meals such as soybean oil meal, rapeseed oil meal,coconut oil meal and linseed oil meal; brans such as wheat bran, ricebran, and defatted rice bran; production meals such as cone gluten mealand corn jam meal; animal or fish-derived feeds such as fish meal, skimmilk powder, whey, yellow grease and tallow; yeasts such as torula yeastand brewer's yeast; mineral material feeds such as tribasic calciumphosphate and calcium carbonate; oils and fats; monomeric amino acids;saccharides, and so forth. Examples of the form of the feed include, forexample, pet food, livestock feed, fish breeding feed, and so forth.

The amount of the extract of Citrus depressa or polymethoxyflavonoidcontained in the food or drink (including feed) of the present inventionis not particularly limited, and may be appropriately chosen. However,for example, when a extract of Citrus depressa is used, the amountthereof is preferably 1 mass % or more in terms of the amount of solidmatter contained in the extract of Citrus depressa. Alternatively, theamount of the extract of Citrus depressa contained in the food or drinkmay be 0.3 mass % or more, preferably 0.6 mass % or more, furtherpreferably 3 mass % or more, particularly preferably 10 mass % or more,in terms of polymethoxyflavonoid content. Although the maximum contentof the extract of Citrus depressa is not particularly limited, it maybe, for example, 95 mass % or less, 50 mass % or less, 30 mass % orless, 20 mass % or less, or 10 mass % or less, in terms of the amount ofsolid matter in the extract of Citrus depressa, or it may be, forexample, 95 mass % or less, 70 mass % or less, 60 mass % or less, 50mass % or less, or 40 mass % or less, in terms of the amount ofpolymethoxyflavonoid.

Further, when the polymethoxyflavonoid is used, the amount ofpolymethoxyflavonoid contained in the food or drink is preferably 0.3mass % or more, more preferably 0.6 mass % or more, still morepreferably 3.0 mass % or more, particularly preferably 10 mass % ormore, in terms of solid matter. Although the maximum content of thepolymethoxyflavonoid is not particularly limited, it may be, forexample, 95 mass % or less, 70 mass % or less, 60 mass % or less, 50mass % or less, or 40 mass % or less.

When nobiletin is used, the amount of nobiletin contained in the food ordrink is preferably 0.2 mass % or more, more preferably 0.4 mass % ormore, still more preferably 2.0 mass % or more, particularly preferably5 mass % or more, in terms of solid matter. Although the maximum contentof nobiletin is not particularly limited, it may be, for example, 95mass % or less, 70 mass % or less, 50 mass % or less, 30 mass % or less,or 10 mass % or less.

When tangeretin is used, the amount of tangeretin contained in the foodor drink is preferably 0.1 mass % or more, more preferably 0.2 mass % ormore, still more preferably 1.0 mass % or more, particularly preferably2 mass % or more, in terms of solid matter. Although the maximum contentof tangeretin is not particularly limited, it may be, for example, 95mass % or less, 70 mass % or less, 50 mass % or less, 30 mass % or less,or 10 mass % or less.

Further, the food or drink (including feed) of the present inventiondesirably contains 5 mg or more, preferably 18 mg or more, morepreferably 180 mg or more, of the extract of Citrus depressa in terms ofsolid matter in an amount for single ingestion.

Further, the food or drink (including feed) of the present inventiondesirably contains 1.8 mg or more, preferably 18 mg or more, morepreferably 180 mg or more, of the polymethoxyflavonoid in terms of solidmatter in an amount for single ingestion.

Further, the food or drink (including feed) of the present inventiondesirably contains 1.2 mg or more, preferably 12 mg or more, morepreferably 120 mg or more, of nobiletin in terms of solid matter in anamount for single ingestion.

Further, the food or drink (including feed) of the present inventiondesirably contains 0.6 mg or more, preferably 6 mg or more, morepreferably 60 mg or more, of tangeretin in terms of solid matter in anamount for single ingestion.

EXAMPLES

Hereafter, the present invention will be more specifically explainedwith reference to examples. However, the present invention is notlimited to these examples.

Example 1

Muscle atrophy inhibition effects of extract of Citrus depressa,polymethoxyflavonoid, nobiletin, and tangeretin were evaluated in aglucocorticoid-induced rat muscle atrophy model.

The extract of Citrus depressa used (commercial product, ARKRAY) wasobtained by adding cyclodextrin as a clathrating agent to an extractfrom squeezed residue of Citrus depressa fruits with water-containingethanol, and had the following composition according to the usualspecification thereof.

Solid matter 92 mass % or more Cyclodextrin 50 mass %Polymethoxyflavonoids 10 mass % or more

Nobiletin content and tangeretin content of the extract of Citrusdepressa (containing cyclodextrin) used for the following experimentwere 6.9 to 8.5 mass % and 3.4 to 4.1 mass %, respectively.

As the glucocorticoid for preparing the muscle atrophy model,dexamethasone was used.

Male SD rats (15-month old) were preliminarily fed for one week, anddivided into three groups (dexamethasone non-administration group (CTL),dexamethasone administration group (DEX), and dexamethasone and extractof Citrus depressa (Shiikuwasha extract) administration group (SE), n=6for the DEX and CTL groups, n=5 for the SE group).

Then, the rats of the DEX group and the CTL group were fed with standardfeed AIN-93M (CLEA Japan), and the rats of the SE group were fed withAIN-93M added with the extract of Citrus depressa at a ratio of 1 mass%, for two weeks.

After two-week feeding, the rats of the DEX group and SE group wereintraperitoneally administered with 750 μg/kg body weight ofdexamethasone once a day for 5 days to induce muscle atrophy. The ratsof the CTL group were intraperitoneally administered with physiologicalsaline once a day for 5 days.

On the 6th day from the start of the administration of dexamethasone,the rats were dissected, the left hindlimb tibialis anterior muscleswere collected, and wet weights of the muscles were measured. The muscleatrophy inhibition effect was evaluated according to differences inweights of the muscles of the groups.

As shown in Table 1, the tibialis anterior muscle weight of thedexamethasone administration group (DEX) significantly and markedlydecreased to about 78.7% of that of the dexamethasone non-administrationgroup (CTL), and thus it was confirmed that the muscles were atrophiedby administration of dexamethasone. Further, the muscle weight of thedexamethasone and extract of Citrus depressa (shiikuwasha extract)administration group (SE) fed with the feed containing the extract ofCitrus depressa was significantly larger than that of the DEX group(about 92.3% of that of the CTL group, p<0.05, statistically significantdifference over the DEX group according to the Dunnett test), and thusit was confirmed that the muscle atrophy was inhibited by the extract ofCitrus depressa.

TABLE 1 CTL DEX SE Wet muscle weight (g) 1.187 0.934 1.096 SD 0.1230.111 0.094

Example 2

The tibialis anterior muscles collected in Example 1 mentioned abovewere used to evaluate expression amounts of genes that participate inmuscle atrophy.

The tibialis anterior muscle was frozen in liquid nitrogen immediatelyafter the collection, and homogenized in Trizol Reagent (Invitrogen),and the total RNA was extracted by using RNeasy Mini Kit (QIAGEN). cDNAwas obtained by the total RNA by using High Capacity cDNA Reverse Kit(ABI). The cDNA, primers for amplifying each of the atrogin-1, MuRF1,and myostatin genes (ABI), and Taqman Fast Universal PCR Master Mix(ABI) were mixed, and expression amount of each gene in the test samplewas relatively quantified by the real time PCR method, with taking thegene expression amount in the DEX group as 1. In statistical analysis,it was determined whether there were statistically significantdifferences between the values obtained for the DEX group and the valuesobtained for the other groups according to the Dunnett test.

It is known that muscle atrophy is accompanied by increased muscleprotein degradation, in addition to reduction of muscle proteinsynthesis. The major protein degradation systems in skeletal musclesinclude three systems, ubiquitin-proteasome system, calpain system, andautophagy system, and it is considered that, among these, theubiquitin-proteasome system plays an especially important role. Proteinsare labeled by ubiquitin ligase, and the ubiquitinated proteins aredegraded by proteasome. It is considered that by inhibiting theubiquitin-proteasome pathway, protein degradation in muscle atrophy canbe inhibited (Tawa, N. E. Jr., J. Clin. Invest., 100(1):197-203, 1997).

As muscle-specific ubiquitin ligases for which it has been to dateelucidated that expression amount of the gene thereof increases at thetime of muscle atrophy, there are known MuRF1 (Nikawa, T. et al., TheFASEB Journal express article 10.1096/fj.03-0419fje, Published onlineJan. 8, 2004), and atrogin-1 (Gomes, M. D. et al., PNAS, 98(25) 2001).Further, myostatin (also called as growth/differentiation factor 8) geneis also known as a repressor of muscle growth (McPherron, A. C. et al.,Nature, 387:83-90, 1997).

The results of analysis of expression amounts of the genes performed byreal-time PCR are shown in FIG. 1.

As for the gene expression amounts in the CTL group, the expressionamounts of the atrogin-1 gene and the MuRF1 gene were 0.26±0.06 and0.10±0.02, respectively, relative to the expression amounts in the DEXgroup, which were taken as 1.0, and thus it was confirmed that they weresignificantly increased by the administration of dexamethasone. Incontrast, in the SE group, the expression amounts of the atrogin-1 geneand the MuRF1 gene were 0.73±0.12 (p<0.01) and 0.54±0.15 (p<0.05),respectively, which were both significantly reduced from those observedin the DEX group, and thus it was confirmed that the expression of themuscle atrophy-related genes was significantly inhibited by theingestion of the extract of Citrus depressa.

Example 3

In Examples 1 and 2, 1 mass % of the extract of Citrus depressa wasadded to the feed, and fed. However, in this example, for individualevaluation of polymethoxyflavonoid, nobiletin (Tokyo Chemical Industry),and tangeretin (Tokyo Chemical Industry), they were each mixed with thefeed at a ratio of 0.001 mass % for polymethoxyflavonoid (PMF group),0.0007 mass % for nobiletin (NOB group), or 0.0004 mass % for tangeretin(TAN group), and fed for two weeks. Then, dexamethasone was administeredto the animals of the groups to induce muscle atrophy. These groups wereexamined by comparison with the non-treatment group (CTL group) and thedexamethasone administration group (DEX group). As thepolymethoxyflavonoid, a mixture of nobiletin and tangeretin at a ratioof 6.9:3.4 was used.

As for the animal species, SD rats of eight-month old were used, and theevaluation was performed with the same experimental method as that usedin Examples 1 and 2. On the 6th day from the start of the administrationof dexamethasone, the left hindlimb tibialis anterior muscles werecollected from the rats.

The results of evaluation of gene expression amounts performed in thesame manner as that of Example 2 from tibialis anterior muscles areshown in FIG. 2. As for the gene expression amounts in the CTL group,the expression amounts of the atrogin-1 gene and the MuRF1 gene were0.25±0.11 and 0.11±0.04, respectively, relative to the expressionamounts observed in the DEX group, which were taken as 1.0, and thus itwas confirmed that they were significantly increased by theadministration of dexamethasone. The expression amount of the myostatingene was 0.81±0.39, and increase of the expression amount of this genewas also confirmed. In contrast, in the PMF group, the expression amountof the myostatin gene was 0.40±0.08, and thus decrease of the expressionamount was observed. In the NOB group, the expression amount of theatrogin-1 gene was 0.85±0.29, and thus decrease of the expression amountwas observed. Also in the TAN group, the expression amount of the MuRF1gene was 0.77±0.35, and thus decrease of the expression amount wasobserved.

Since the average daily Food intake of the rats used for the experimentwas 20.1 g, the polymethoxyflavonoid ingestion amount in the PMF groupwas about 0.20 mg per day. Further, the daily food intake per bodyweight is calculated to be 0.339 mg/l kg of body weight from the averagebody weight (592.7 g). Further, in the NOB group, the nobiletiningestion amount was about 0.14 mg per day, and the daily ingestionamount per unit body weight was about 0.237 mg/l kg of body weight. Inthe TAN group, the daily tangeretin ingestion amount was about 0.08 mg,and the daily ingestion amount per unit body weight was about 0.136 mg/lkg of body weight. From these data, it is considered that ingestionamounts with which the effect can be expected are about 0.3 mg/kg/dayfor polymethoxyflavonoids, about 0.2 mg/kg/day for nobiletin, and about0.1 mg/kg/day for tangeretin.

Further, if it is taken into consideration that the evaluation of thisexperiment was performed with a very short ingestion period of 19 days,it can be expected that, even with a smaller ingestion amount, the sameeffect can be obtained by continuous ingestion.

Example 4

Male SD rats (18-month old) were preliminarily fed for one week, anddivided into three groups (control group (CTL), hindlimb immobilizationgroup (FIX), and hindlimb immobilization and extract of Citrus depressa(shiikuwasha extract) administration group (SE), n=7 for the FIX and CTLgroups, n=6 for the SE group).

Then, the rats of the FIX group and the CTL group were fed with thestandard feed AIN-93M, and the rats of the SE group were fed withAIN-93M added with the extract of Citrus depressa at a ratio of 1 mass%, for two weeks.

After two-week feeding, both hindlimbs of the rats of the FIX group andSE group were immobilized with surgical cast for one week to inducemuscle atrophy. Hindlimbs of the rats of the CTL group were notimmobilized with surgical cast.

On the 8th day from the start of the immobilization of the hindlimbswith surgical cast, the rats were dissected, hindlimb soleus muscleswere collected, and weights of the muscles were measured. In statisticalanalysis, it was determined whether there were statistically significantdifferences between the values obtained for the FIX group and the valuesobtained for the other groups according to the Dunnett test.

The results are shown in FIG. 3. Both the body weight and soleus muscleweight were decreased by immobilization of the hindlimbs with surgicalcast. The soleus muscle weight per kg of body weight was 0.37±0.03 forthe CTL group, whereas the same observe for the FIX group was 0.33±0.04,and thus decreased compared with that observed for the CTL group.However, in the SE group, the soleus muscle weight was 0.39±0.07, thussignificantly increased compared with the FIX group (p<0.05), andincreased to the same level as that of the CTL group or further higherlevel.

Example 5 Jelly Food

Among the raw materials mentioned below, nobiletin and cyclodextrin wereused to prepare a solution of nobiletin clathrated with cyclodextrin,and this nobiletin solution and the other raw materials mentioned belowwere dissolved in water to prepare a jelly raw material dissolvedsolution. The solution was sterilized in a conventional manner, andfilled in a cup in an amount of 100 g to prepare jelly food (100 g perpiece) having the following composition in a conventional manner. Thecontent of nobiletin in one piece of the obtained jelly food was 70 mg.It was revealed that results indicating muscle atrophy inhibition actioncould be obtained by ingesting two pieces per day of this food for along period of time.

dextrin (Matsutani Chemical Industry) 25.0 mass % Whey protein (MorinagaMilk Industry) 12.5 mass % gelling agent (San-Ei Gen F.F.I.) 0.3 mass %citric acid (San-Ei Gen F.F.I.) 0.2 mass % ascorbate Na (DSM Nutrition)0.1 mass % nobiletin (Tokyo Chemical Industry) 0.07 mass % cyclodextrin(San-Ei Gen F.F.I.) 0.07 mass % flavor (San-Ei Gen F.F.I.) 0.02 mass %vitamin D (San-Ei Gen F.F.I.) 5.0 × 10⁻⁷ mass % water 61.74 mass %

Example 6 Drink

Among the raw materials mentioned below, tangeretin and cyclodextrinwere used to prepare a solution of tangeretin clathrated withcyclodextrin, and this tangeretin solution and the other raw materialsmentioned below were dissolved in water to prepare a drink raw materialdissolved solution. The solution was filled in a bottle to prepare adrink (500 ml per bottle) having the following composition in aconventional manner. The content of tangeretin in the obtained drinkcontained in one container was 55.51 mg. It was revealed that resultsindicating muscle atrophy inhibition action could be obtained byingesting two containers per day of this drink for a long period oftime.

dextrin (Matsutani Chemical Industry) 7.0 mass % protein hydrolysate 0.5mass % (Morinaga Milk Industry) citric acid (San-Ei Gen F.F.I.) 0.2 mass% ascorbate Na (DSM Nutrition) 0.2 mass % flavor (San-Ei Gen F.F.I.)0.02 mass %  sweetener (San-Ei Gen F.F.I.) 0.01 mass %  tangeretin(Tokyo Chemical Industry) 0.008 mass %  cyclodextrin (San-Ei Gen F.F.I.)0.008 mass %  water 92.05 mass % 

Example 7 Tablet Confectionary

A mixture having the following composition was tableted in aconventional manner to produce tablet confectionaries having a weight of250 mg per tablet. Content of the extract of Citrus depressa in 1 g ofthe obtained tablet confectionaries was 60 mg. Since thepolymethoxyflavonoid content in the extract of Citrus depressa used asthe raw material was 10% or higher, content of the polymethoxyflavonoidin 1 g of the tablet confectionaries was about 6 mg. It was revealedthat results indicating muscle atrophy inhibition action could beobtained by ingesting 16 tablets per day of this food for a long periodof time.

powder candy (Showa Sangyo) 86.0 mass %  extract of Citrus depressa(Arkray) 6.0 mass % citric acid (San-Ei Gen F.F.I.) 4.0 mass % flavor(San-Ei Gen F.F.I.) 2.0 mass % emulsifier (Kao) 2.0 mass %

Example 8 Chewable Tablet

Chewable tablets having the following composition and weight of 250 mgper tablet were produced in a conventional manner. Content of theextract of Citrus depressa in 1 g of the obtained chewable tablets was200 mg. Since the polymethoxyflavonoid content in the extract of Citrusdepressa used as the raw material was 10% or higher, content of thepolymethoxyflavonoid in 1 g of the chewable tablets was about 20 mg. Itwas revealed that results indicating muscle atrophy inhibition actioncould be obtained by ingesting 4 tablets per day of this food for a longperiod of time.

erythritol (Mitsubishi Chemical Foods) 68.0 mass %  extract of Citrusdepressa (Arkray) 20.0 mass %  citric acid (San-Ei Gen F.F.I.) 7.0 mass% talc (San-Ei Gen F.F.I.) 3.0 mass % flavor (San-Ei Gen F.F.I.) 2.0mass %

Example 9 Enteral Nutrient (Concentrate Liquid Diet)

Casein and hardly digestible dextrin were dissolved in warm water, thendextrin, a mineral mixture, a vitamin mixture, and nobiletin clathratedwith cyclodextrin were mixed with the solution, an emulsifier andsoybean oil were added to the mixture, and the mixture was homogenized.The mixture was sterilized and filled in a conventional manner toprepare an enteral nutrient having the following composition. Themineral mixture and vitamin mixture mentioned below were obtained bymixing the ingredients in the amounts shown in Table 2. Content ofnobiletin in 1000 ml of the obtained enteral nutrient was 46 mg. It wasrevealed that results indicating muscle atrophy inhibition action couldbe obtained by ingesting 1000 ml per day of this food for a long periodof time.

dextrin (Matsutani Chemical Industry) 15.0 mass %  casein sodium(Morinaga Milk Industry) 4.0 mass % soybean oil (Taiyo Yushi) 3.0 mass %hardly digestible dextrin 1.0 mass % (Matsutani Chemical Industry)mineral mixture 0.3 mass % emulsifier (San-Ei Gen F.F.I.) 0.05 mass % vitamin mixture 0.02 mass %  flavor (San-Ei Gen F.F.I.) 0.01 mass % nobiletin (Tokyo Chemical Industry) 0.0046 mass %   cyclodextrin (San-EiGen F.F.I.) 0.0046 mass %   water 76.6108 mass %  

TABLE 2 (/1000 ml) mineral mixture Na 900 mg K 1500 mg Ca 750 mg Mg 380mg Fe 11 mg vitamin mixture β-carotene 1800 μg Vitamin D 5 μgα-tocopherol 12 mg vitamin B1 1.6 mg vitamin B2 1.8 mg vitamin B6 3 mgvitamin B12 3 μg vitamin C 100 mg

INDUSTRIAL APPLICABILITY

According to the present invention, a muscle atrophy inhibitor isprovided. The muscle atrophy inhibitor of the present invention can beused as a drug. Further, since the muscle atrophy inhibitor of thepresent invention uses an ingredient contained in Citrus depressa as theactive ingredient, it is highly safe, and can be used for foods, drinks,and so forth.

What is claimed is:
 1. A method for inhibiting muscle atrophy, whichcomprises administering a polymethoxyflavonoid to a mammal in atherapeutically effective amount.
 2. The method for inhibiting muscleatrophy according to claim 1, wherein the amount of polymethoxyflavonoidis 0.03 mg/kg/day or more and 150 mg/kg/day or less.
 3. The method forinhibiting muscle atrophy according to claim 1, wherein thepolymethoxyflavonoid comprises nobiletin and/or tangeretin.
 4. Themethod for inhibiting muscle atrophy according to claim 1, wherein themuscle atrophy is caused by aging, bedridden, sedentary lifestyle,spaceflight, immobilization of limbs performed for treatment of injury,postoperative rest, side reaction of a drug, paralysis, spinal cordinjury, traumatic injury of peripheral nerve, osteoarthritis, rheumatoidarthritis, diabetes, thermal burn, polio, Guillain-Barre syndrome,muscular dystrophy, congenital myotonia, infectious disease accompaniedby inflammation, sepsis accompanying infectious disease, inflammatorybowel disease, connective tissue disease, renal failure, hepaticfailure, cardiac failure, cancer, malignant tumor, cachexia, anorexia orhypercatabolism in terminal symptoms of a disease, or amyotrophiclateral sclerosis.
 5. The method according to claim 4, wherein the causeis a side reaction of a drug and wherein the drug is a steroid.
 6. Themethod according to claim 4, wherein the cause is infectious diseaseaccompanied by inflammation and wherein the infectious disease is AIDSor viral hepatitis.